Bioluminescence method utilizing bioluminescence produced by a combination of firefly luciferase and a luciferin does not require excitation light, and accordingly produces substantially no background noise to achieve highly sensitive measurement. Further, since the method does not require excitation light, measurements can be conducted by using an apparatus with a simple structure. Due to these characteristic features, the bioluminescence method is widely used to detect target substances in blotting, reporter enzymes for various assay systems and the like. Furthermore, the method also enables imaging of deep portions of a biological sample having poor light transmission, and therefore, applications of the method to in vivo imaging systems have recently been being studied.
The bioluminescence method is believed to be effective in in viva imaging as described above. However, since the emission wavelength in the conventional bioluminescence method is limited to 570 nm as that of luciferin, a problem arises that the wavelength is too short for use in in vivo imaging. Therefore, researches have been made to shift the emission wavelength to a longer side to achieve superior tissue transmission. However, most of these researches have been conducted by modifying characteristic features of luciferase as an enzyme, so there is almost no report whose approach is to modify luciferin as substrates.
On the other hand, although sensitivity of the bioluminescence method have been mainly focused on, some reports are directed to studies on functional luciferase substrates that enable on/off control of the bioluminescent feature so that luminescence is generated only in the presence of a target molecule (for example, Japanese Patent Laid-Open Publication (Kokai) No. 2000-270894; Miska, W., Geiger, R., J. Clin. Chem. Clin. Biochem., January; 25(1) pp.23-30, 1987, etc.). These studies relate to on/off control type luminescent probes using luciferin and analogues thereof, which utilize the feature that methyl ether-type luciferin or amide-type aminoluciferin does not show luminescence, and are based on the principle of detecting activity of an enzyme that specifically reacts with a sugar or a peptide that binds to these luciferin analogues beforehand. However, this principle has a problem that applicable targets of probes are very limited. Therefore, it has been desired to develop a widely applicable bioluminescent probe that enables functional luminescence imaging in vivo.